The long term goal of this research is to understand how multiple carcinogens interact with cells to produce cancer. Agreat deal of work has been done on the effect of individual carcinogens, but to better mimic the actual environment to which humans are exposed, studies must be performed examining the effect of multiple carcinogen exposure. This proposal will specifically examine the interaction of metals (chromium and nickel) and benz[a]alpyrene diol epoxide (BPDE), all common environmental carcinogens, on the mutant frequency of the hypoxanthine phosphoribosyltransferase (HPRT) gene in normal human fibroblasts. Previous work in applicant's laboratory has shown that normal human fibroblasts treated with either potassium dichromate or nickel subsulfide are refractive to the mutagenic effects of BPDE. This antagonistic effect can be inhibited if antioxidants are included in the cell medium during the treatment period, suggesting a role for reactive oxygen species. This proposal will examine, in more detail, the biochemical mechanism for this antagonistic effect. Two hypothesis will be directly tested. The first is that low concentrations of metals can act in a synergistic way with BPDE to induce the activity of DNA repair enzymes. This hypothesis will be tested by directly measuring the DNA repair capability of cells treated with chromium or nickel and BPDE with untreated cells or cells treated with only one of the carcinogens. This hypothesis will be further tested by repeating experiments which showed the antagonistic effect with repair deficient cell lines. The antagonistic effect should not be observed in these cell lines if DNA repair processes are responsible for the antagonistic effect. The second hypothesis that will be tested is that low concentrations of metals can act synergistically with BPDE to cause inhibition of cell cycle progression. This could cause a reduction in the mutant frequency of BPDE by giving the cell more time to repair damaged DNA prior to DNA replication. Possible effects on the cell cycle will be tested by directly measuring the progression of cells through G and into S phase in the presence of metal and BPDE, and comparing that to untreated cells and cells treated with only one of the carcinogens. In addition, expression of proteins known to inhibit cell cycle progression from G to S phase will be determined, and the effect of plasmids carrying dominant-negative mutants of these cell cycle regulatory proteins will be examined.